The invention relates generally to methods for identifying agents that affect mitochondrial membrane permeability transition. More specifically, the invention relates to compositions and screening methods for use in identifying agents that alter the interaction between the mitochondrial adenine nucleotide translocator and cyclophilin D.
Mitochondria are the main energy source in cells of higher organisms, and provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes. Such processes include electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which also underlies a central mitochondrial role in intracellular calcium homeostasis.
Mitochondrial ultrastructural characterization reveals the presence of an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes. The subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix. (For a review, see, e.g., Emster et al., 1981 J. Cell Biol. 91:227s.) The cristae, originally postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tube-like conduits that may form networks, and that can be connected to the inner membrane by open, circular (30 nm diameter) junctions (Perkins et al., 1997, Journal of Structural Biology 119:260). While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large ( greater than xcx9c10 kDa) molecules.
Altered or defective mitochondrial activity, including but not limited to failure at any step of the ETC, may result in catastrophic mitochondrial collapse that has been termed xe2x80x9cpermeability transitionxe2x80x9d (PT) or xe2x80x9cmitochondrial permeability transitionxe2x80x9d (MPT). According to generally accepted theories of mitochondrial function, proper ETC respiratory activity requires maintenance of an electrochemical potential (xcex94xcexa8m) in the inner mitochondrial membrane by a coupled chemiosmotic mechanism. Altered or defective mitochondrial activity may dissipate this membrane potential, thereby preventing ATP biosynthesis and halting the production of a vital biochemical energy source. In addition, mitochondrial proteins such as cytochrome c may leak out of the mitochondria after permeability transition and may induce the genetically programmed cell suicide sequence known as apoptosis or programmed cell death (PCD).
Four of the five multi-subunit protein complexes (Complexes I, III, IV and V) that mediate ETC activity are localized to the inner mitochondrial membrane, which is the most protein rich of biological membranes in cells (75% by weight); the remaining ETC complex (Complex II) is situated in the matrix. In at least three distinct chemical reactions known to take place within the ETC, positively-charged protons are moved from the mitochondrial matrix, across the inner membrane, to the intermembrane space. This disequilibrium of charged species creates an electrochemical potential of approximately 220 mV referred to as the xe2x80x9cproton motive forcexe2x80x9d (PMF), which is often represented by the notation xcex94"psgr" or xcex94"psgr"m and represents the sum of the electric potential and the pH differential across the inner mitochondrial membrane (see, e.g., Emster et al., 1981 J. Cell Biol. 91:227s and references cited therein).
This membrane potential provides the energy contributed to the phosphate bond created when adenosine diphosphate (ADP) is phosphorylated to yield ATP by ETC Complex V, a process that is xe2x80x9ccoupledxe2x80x9d stoichiometrically with transport of a proton into the matrix; xcex94"psgr"m is also the driving force for the influx of cytosolic Ca2+ into the mitochondrion. Under normal metabolic conditions, the inner membrane is largely impermeable to proton movement from the intermembrane space into the matrix, leaving ETC Complex V as the primary means whereby protons can return to the matrix. When, however, the integrity of the inner mitochondrial membrane is compromised, as occurs during MPT that may accompany a disease associated with altered mitochondrial function, protons are able to bypass the conduit of Complex V without generating ATP, thereby xe2x80x9cuncouplingxe2x80x9d respiration because electron transfer and associated proton pumping yields no ATP. Thus, mitochondrial permeability transition involves the opening of a mitochondrial membrane xe2x80x9cporexe2x80x9d, a process by which, inter alia, the ETC and xcex94"psgr"m are uncoupled, xcex94"psgr"m collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small (e.g., ionic Ca2+, Na+, K+, H+) and large (e.g., proteins).
The mitochondrial permeability transition xe2x80x9cporexe2x80x9d may not be a discrete assembly or multi-subunit complex, and the term thus refers instead to any mitochondrial molecular component (including, e.g., a mitochondrial membrane per se) that regulates the inner membrane selective permeability where such regulated function is impaired during MPT. A mitochondrial molecular component may be a protein, polypeptide, peptide, amino acid or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof; a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like; or any other biological molecule that is a constituent of a mitochondrion. A mitochondrial permeability transition pore component, also referred to as a mitochondrial pore component, may be any mitochondrial molecular component that regulates the selective permeability characteristic of mitochondrial membranes as described above, including those responsible for establishing xcex94xcexa8m and those that are functionally modified during MPT. Mitochondrial pore components may also include factors that interact with mitochondria, for example through transient or stable association with other mitochondrial pore components, in a manner that regulates MPT. Examples of such factors include cyclophilins (described in greater detail below), calcium modulating cyclophilin ligand (CAML, see, e.g., Table 1, infra, and references cited therein) and members of the Bcl-2 family including Bcl-2 (e.g., Green et al., 1998 Science 281:1309), Bax (Marzo et al., 1998 Science 281:2027) and Bak (Narita et al., 1998 Proc. Nat. Acad. Sci. USA 95:14681).
Without wishing to be bound by theory, it is unresolved whether this pore is a physically discrete conduit that is formed in mitochondrial membranes, for example by assembly or aggregation of particular mitochondrial and/or cytosolic proteins and possibly other molecular species, or whether the opening of the xe2x80x9cporexe2x80x9d may simply represent a general increase in the porosity of the mitochondrial membrane. In any event, certain mitochondrial molecular components may contribute to the MPT mechanism, including ETC components or other mitochondrial components described herein. For example, some non-limiting examples of mitochondrial permeability transition pore components that appear to contribute to the MPT mechanism include members of the following families of gene products (see, e.g., Table 1, infra, and references cited therein): adenine nucleotide translocator (ANT); peripheral benzodiazepine receptor (PBzR; McEnery et al., 1992 Proc. Nat. Acad. USA 89:3170); PBzR-associated protein (PRAX); voltage dependent anion channel (VDAC, also known as porin); cyclophilin (Cyp); calcium modulating cyclophilin ligand (CAML); the mitochondrial calcium uniporter, mitochondria associated hexokinase(s) and mitochondrial intermembrane creatine kinases.
MPT may result from direct or indirect effects of mitochondrial genes, gene products or downstream mediator molecules and/or extramitochondrial genes, gene products or downstream mediators. MPT may also result from other known or unknown causes. Loss of mitochondrial potential may be a critical event in the progression of diseases associated with altered mitochondrial function, including degenerative diseases.
Mitochondrial defects may contribute significantly to the pathogenesis of diseases associated with altered mitochondrial function. Such defects may be related to the discrete mitochondrial genome that resides in mitochondrial DNA (i.e., the mitochondrial chromosome) and/or to the extramitochondrial genome, which includes nuclear chromosomal DNA and other extramitochondrial DNA, For example, alterations in the structural and/or functional properties of mitochondrial components, including alterations deriving from genetic and/or environmental factors or alterations derived from cellular compensatory mechanisms, may play a role in the pathogenesis of any disease associated with altered mitochondrial function. A number of degenerative diseases are thought to be caused by, or to be associated with, alterations in mitochondrial function. These include Alzheimer""s Disease (AD); diabetes mellitus; Parkinson""s Disease; Huntington""s disease; dystonia; Leber""s hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome. The extensive list of additional diseases associated with altered mitochondrial function continues to expand as aberrant mitochondrial or mitonuclear activities are implicated in particular disease processes.
A hallmark pathology of AD and potentially other diseases associated with altered mitochondrial function is the death of selected cellular populations in particular affected tissues, which results from apoptosis (also referred to as xe2x80x9cprogrammed cell deathxe2x80x9d or PCD). Mitochondrial dysfunction is thought to be critical in the cascade of events leading to apoptosis in various cell types (Kroemer et al., FASEB J. 9:1277-87, 1995), and may be a cause of apoptotic cell death in neurons of the AD brain. Altered mitochondrial physiology may be among the earliest events in PCD (Zamzami et al., J. Exp. Med. 182:367-77, 1995; Zamzami et al., J. Exp. Med. 181:1661-72, 1995) and elevated reactive oxygen species (ROS) levels that result from such altered mitochondrial function may initiate the apoptotic cascade (Ausserer et al., Mol. Cell. Biol. 14:5032-42, 1994).
Thus, in addition to their role in energy production in growing cells, mitochondria (or, at least, mitochondrial components) participate in apoptosis (Newmeyer et al., 1994, Cell 79:353-364; Liu et al., 1996, Cell 86:147-157). Apoptosis is apparently also required for, inter alia, normal development of the nervous system and proper functioning of the immune system. Moreover, some disease states are thought to be associated with either insufficient (e.g., cancer, autoimmune diseases) or excessive (e.g., stroke damage, AD-associated neurodegeneration) levels of apoptosis. For general reviews of apoptosis, and the role of mitochondria therein, see Green and Reed (1998, Science 281:1309-1312), Green (1998, Cell 94:695-698) and Kromer (1997, Nature Medicine 3:614-620). Hence, agents that affect apoptotic events, including those associated with mitochondrial components, might have a variety of palliative, prophylactic and therapeutic uses.
The adenine nucleotide translocator (ANT) is an example of one particular mitochondrial pore component as provided herein. ANT, nuclear encoded polypeptide that is a major component of the inner mitochondrial membrane, is responsible for mediating transport of ADP and ATP across the mitochondrial inner membrane. For example, ANT is believed to mediate stoichiometric ATP/proton exchange or cotransport across the inner mitochondrial membrane, and ANT inhibitors (such as atractyloside or bongkrekic acid) induce MPT under certain conditions. Three human ANT isoforms have been described that differ in their tissue expression patterns and other mammalian ANT homologues have been described (see, e.g., Wallace et al., 1998 in Mitochondria and Free Radicals in Neurodegenerative Diseases, Beal, Howell and Bodis-Wollner, Eds., Wiley-Liss, New York, pp. 283-307, and references cited therein). ANT has also been implicated as an important molecular component of the mitochondrial permeability transition pore, a Ca2+-regulated inner membrane channel that, as described above, plays an important modulating role in apoptotic processes.
Members of the cyclophilin family of highly conserved proteins provide an example of mitochondrial pore components as provided herein that are factors which, as described above, may stably or transiently interact with other mitochondrial pore components in a manner that regulates MPT. The cyclophilins (Cyps) are a family of ubiquitous proteins expressed in all organisms. All Cyp family members share a conserved core of about 109 amino acids, but differ from one another by unique extensions that function in organelle and membrane transport (e.g., Walsh et al., 1992 J. Biol. Chem. 267:13115-18). At least eight human Cyp isoforms are known, including single domain and two-domain cyclophilins (e.g., Taylor et al., 1997 Prog. Biophys. Mol. Biol. 67:155-81). Distinct isoforms localize to different cell compartments, including cytoplasmic, endoplasmic reticulum (ER), mitochondrial, and cell surface isoforms (Handler et al. EMBO J. 6: 947-50, 1987; Price et al. Proc. Natl. Acad. Sci. USA 88: 1903-07, 1991; Bergsma et al. J. Biol. Chem. 266: 23204-14; Cacalano et al. Proc Natl Acad Sci USA 89: 4353-57, 1992). For example, and as described in greater detail below, Cyclophilin D (CypD) is another molecule that may regulate mitochondrial permeability.
Cyclophilins are believed to perform multiple functions within cells. For example, Cyps catalyze the interconversion of cis and trans isomers of peptidylprolyl bonds in peptides and proteins, thereby facilitating the folding of proteins for which isomerization of peptidylprolyl bonds is rate limiting (see, e.g., Galat, Eur. J. Biochem. 216:689-707, 1993; Fischer et al., Biochem. 29:2205-2212, 1990; Stamnes et al., Cell 65:219-27, 1991). This peptidylproyl cis-trans-isomerase activity can be blocked by the immunosuppressant cyclosporin A (e.g., Fruman et al., Proc. Natl. Acad. Sci. USA 89:3741-45, 1992). Cyp family members also appear to mediate other activities by forming complexes with fully folded, functional proteins (see, e.g., Jaschke et al., J. Mol. Biol. 277:763-69, 1998; Ratajczk et al., J. Biol. Chem. 268:13187-92, 1993; Wu et al., J. Biol. Chem. 270:14209-19, 1995; Holloway et al., J. Biol. Chem. 273:16346-50, 1998; Franke et al., Adv. Exp. Med. Biol. 374: 217-28, 1995).
CypD is the only mitochondrial isoform of the Cyp family identified to date. The human CypD polypeptide is 207 amino acids long and has an NH2-terminal hydrophobic extension, which may serve to transport the polypeptide across mitochondrial membranes to the matrix (Bergsma et al., J. Biol. Chem. 266:23204-14, 1991). Cyp D is believed to participate in the formation of the mitochondrial permeability transition pore by interacting with the voltage-dependent anion channel (VDAC) and with ANT, at contact sites between the mitochondrial outer and inner membranes (Crompton et al., Eur. J. Biochem. 258 729-35, 1998; Woodfield et al., 1998, Biochem. J. 336:287-90). CypD binding to ANT may also sensitize the pore complex to Ca2+ concentration (Halestrap et al., Biochim. Biophys. Acta. 1366:79-94, 1998). In vitro, relatively high Ca2+ concentrations increase mitochondrial membrane permeability, resulting in free diffusion of low molecular weight solutes across the inner membrane (e.g., Halestrap et al., Mol. Cell. Biochem. 174:167-172, 1997; Hunter et al., Arch. Biochem. Biophys. 195:453-59, 1979). Oxidative stress, adenine nucleotide depletion and decreased membrane potential may also increase mitochondrial permeability (e.g., Bernardi et al., J. Bioenerg. Biomembr. 26:509-17, 1994; Zoratti et al., Biochim. Biophys. Acta 1241:139-76, 1995). This opening of the mitochondrial permeability transition pore may be an event in the pathogenesis of diseases associated with altered mitochondrial function, such as those described above. For example, MPT may contribute to necrotic cell death following vascular ischemia/reperfusion injury as may occur following cardiac bypass surgery, thrombolysis and organ transplantation (e.g., Halestrap et al., Biochem. Soc. Trans. 21:353-58, 1993; Halestrap et al., Mol. Cell. Biochem. 174:167-172, 1997). As another example, a VDAC-ANT-CypD complex may also participate in mitochondrial outer membrane rupture resulting in the release of apoptogenic proteins from the intermembrane space (e.g., Petit et al., FEBS Lett. 426:111-16, 1998; Marzo et al. J. Exp. Med. 187:1261-71, 1998).
To provide improved therapies for diseases associated with altered mitochondrial function such as those discussed above, agents that alter mitochondrial permeability transition may be beneficial, and assays to specifically detect such agents are needed. The present invention fulfills these needs and further provides other related advantages.
The present invention is directed to assays for identifying and using agents that alter mitochondrial membrane permeability transition, and to related compositions and methods. In one aspect, the invention provides a nucleic acid expression construct comprising a promoter operably linked to a polynucleotide encoding a mitochondrial permeability transition pore component polypeptide fused to an energy transfer molecule polypeptide, or a variant thereof. In one embodiment the mitochondrial permeability transition pore component is an adenine nucleotide translocator, which in certain further embodiments is human ANT1, human ANT2 or human ANT3. In one embodiment the mitochondrial permeability transition pore component is porin, hexokinase, creatine kinase, PRAX, CAML or the peripheral benzodiazepine receptor. The invention also provides, in certain embodiments, a nucleic acid expression construct comprising a promoter operably linked to a polynucleotide encoding a cyclophilin polypeptide fused to an energy transfer molecule polypeptide, or a variant thereof. In one embodiment the cyclophilin is cyclophilin D, and in other embodiments the cyclophilin is human cyclophilin A, cyclophilin B, human cyclophilin C or human Cyp-60. In certain embodiments the expression construct comprises a vector that is a plasmid, a cosmid, a shuttle vector, a viral vector or a vector comprising a chromosomal origin of replication. In certain embodiments the vector comprises a plasmid that is pBAD-His, pEYFP-C1 or pECFP-N1.
According to certain embodiments of the invention, the promoter is externally regulated. In some embodiments the energy transfer molecule is a green fluorescent protein (GFP), a FLASH sequence or an aequorin protein. In certain further embodiments the green fluorescent protein is blue-shifted GFP, cyan-shifted GFP, red-shifted GFP or yellow-shifted GFP. In certain other embodiments the energy transfer molecule is a derivative of an energy transfer molecule selected that is a green fluorescent protein (GFP), a FLASH sequence or an aequorin protein.
In another aspect, the invention provides a polypeptide comprising a mitochondrial permeability transition pore component polypeptide fused to an energy transfer molecule polypeptide, or a derivative thereof. In certain embodiments the mitochondrial permeability transition pore component is an adenine nucleotide translocator, which in certain further embodiments is human ANT1, human ANT2 or human ANT3. In certain other embodiments the mitochondrial permeability transition pore component is porin, hexokinase, creatine kinase, PRAX, CAML or the peripheral benzodiazepine receptor. In another embodiment the invention provides a polypeptide comprising a cyclophilin polypeptide fused to an energy transfer molecule polypeptide, or a derivative thereof. In certain embodiments the cyclophilin is cyclophilin D, and in certain other embodiments the cyclophilin is human cyclophilin A, cyclophilin B, human cyclophilin C or human Cyp-60. In certain embodiments the energy transfer molecule is a green fluorescent protein (GFP), a FLASH sequence or an aequorin protein. In certain further embodiments, the green fluorescent protein is blue-shifted GFP, cyan-shifted GFP, red-shifted GFP or yellow-shifted GFP.
Turning to another aspect, the invention provides a host cell for identifying agents that alter mitochondrial permeability transition, comprising (a) a first nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding a mitochondrial permeability transition pore component polypeptide fused to a polynucleotide encoding a first energy transfer molecule or a variant thereof; and (b) a second nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding a cyclophilin polypeptide fused to a polynucleotide encoding a second energy transfer molecule or a variant thereof, wherein binding of the mitochondrial permeability transition pore component polypeptide to the cyclophilin polypeptide results in detectable energy transfer between the first and second energy transfer molecules. In certain embodiments the mitochondrial permeability transition pore component is an adenine nucleotide translocator, which in certain further embodiments is human ANT1, human ANT2 or human ANT3. In certain other embodiments the mitochondrial permeability transition pore component is porin, hexokinase, creatine kinase, PRAX, CAML or the peripheral benzodiazepine receptor. In certain embodiments the cyclophilin is human cyclophilin A, cyclophilin B, human cyclophilin C or human Cyp-60. In certain other embodiments the host cell is a prokaryotic cell, and in certain other embodiments the host cell is a eukaryotic cell. In certain further embodiments the eukaryotic cell is a 293, COS-7, Sf9, CHO, Hep-2, MDCK or Jurkat cell. In certain other embodiments the first and second energy transfer molecules are green fluorescent protein (GFP), blue-shifted GFP, cyan-shifted GFP, red-shifted GFP or yellow-shifted GFP. In certain other embodiments the first and second energy transfer molecules have an excitation maximum at a wavelength ranging from 300 nm to 650 nm, and an emission maximum at a wavelength ranging from 350 nm to 675 nm. In certain other embodiments the first energy transfer molecule and the second energy transfer molecule have excitation and emission maxima at different wavelengths. In still other certain embodiments at least one nucleic acid expression construct is extrachromosomal, while in other embodiments at least one nucleic acid expression construct is integrated into a host cell chromosome. In certain further embodiments the host cell chromosome is a mitochondrial chromosome.
It is yet another aspect of the invention to provide a method for screening for an agent that alters mitochondrial permeability transition (MPT), comprising the steps of (a) contacting a host cell, according to the invention as described above, comprising a mitochondrion with a candidate agent and an inducer of MPT; (b) exposing the cell to an excitation energy; (c)detecting a level of energy transfer between the first and second energy transfer molecules; and (d) comparing the level of energy transfer to a first reference level generated in the absence of candidate agent, and therefrom identifying an agent that alters MPT. In one embodiment the host cell is further contacted with an inhibitor of MPT to generate a second reference level, and in a further embodiment the inhibitor of MPT is low pH, inducers of high mitochondrial membrane potential or cyclosporin A. In another embodiment the inducer of MPT is atractyloside or bonkrekic acid. In another embodiment the inducer of MPT comprises a compound that increases Ca2+concentration in the mitochondria, and in certain further embodiments the compound is an ionophores, ionomycin, thapsigargin, amino acid neurotransmitters, glutamate, N-methyl-D-aspartic acid, carbachol, apoptogens, or an inducer of potassium depolarization. In another embodiment, the host cell is further contacted with an inducer of oxidative stress, and in certain further embodiments the inducer of oxidative stress is ethacrynic acid, buthionine sulfoximine, diamide, menadione, t-butyl hydroperoxide, phenyl-arsine oxide or nitric oxide. In certain other embodiments the candidate agent increases energy transfer between the first and second energy transfer molecules, while in certain other embodiments the candidate agent decreases energy transfer between the first and second energy transfer molecules. In another embodiment the first and second energy transfer molecules are green fluorescent protein (GFP), blue-shifted GFP, cyan-shifted GFP, red-shifted GFP or yellow-shifted GFP. In some embodiments the excitation energy is light with a wavelength ranging from 300 nm to 650 nm. In other embodiments the first and second energy transfer molecules have an excitation maximum at a wavelength ranging from 300 nm to 650 nm, and an emission maximum at a wavelength ranging from 350 nm to 675 nm.
In certain other embodiments the first energy transfer molecule and the second energy transfer molecule have excitation and emission maxima at different wavelengths. In certain other embodiments (a) the first energy transfer molecule has an excitation maximum at a wavelength ranging from 400 nm to 500 nm and an emission maximum at a wavelength ranging from 450 nm to 525 nm, and the second energy transfer molecule has an excitation maximum at a wavelength ranging from 450 nm to 525 nm and an emission maximum at a wavelength ranging from 500 nm to 550 nm; or (b) the second energy transfer molecule has an excitation maximum at a wavelength ranging from 400 nm to 450 nm and an emission maximum at a wavelength ranging from 450 nm to 500 nm, and the first energy transfer molecule has an excitation maximum at a wavelength ranging from 500 nm to 525 nm and an emission maximum at a wavelength ranging from 525 nm to 550 nm. In certain other embodiments (a) the first energy transfer molecule has an excitation maximum at a wavelength of about 433 nm and an emission maximum at a wavelength of about 475 nm, and the second energy transfer molecule has an excitation maximum at a wavelength of about 513 nm and an emission maximum at a wavelength of about 527 nm; or (b the second energy transfer molecule has an excitation maximum at a wavelength of about 433 nm and an emission maximum at a wavelength of about 475 nm, and the first energy transfer molecule has an excitation maximum at a wavelength of about 513 nm and an emission maximum at a wavelength of about 527 nm.
The present invention provides, in another aspect, a method for detecting an agent that alters mitochondrial permeability transition (MPT), comprising the steps of (a) contacting a cyclophilin D polypeptide with an adenine nucleotide translocator polypeptide and a candidate agent, under conditions and for a time sufficient to permit the cyclophilin D, adenine nucleotide translocator, and the candidate agent to interact; and (b) detecting a level of binding of cyclophilin D polypeptide to adenine nucleotide translocator polypeptide, relative to a level of binding detected in the absence of the candidate agent, and therefrom detecting an agent that alters MPT. In certain embodiments the cyclophilin D polypeptide is immobilized on a support, and in certain embodiments the cyclophilin D polypeptide is a fusion protein. In certain other embodiments the adenine nucleotide translocator polypeptide is immobilized on a support, and in certain embodiments the adenine nucleotide translocator polypeptide is a fusion protein. In certain further embodiments the fusion protein comprises a protease recognition sequence, while in certain other further embodiments the fusion protein comprises a ligand for a receptor. In certain other embodiments the candidate agent is a peptide, a polypeptide, a protein or a small molecule. In some embodiments the candidate agent is a small molecule present within a combinatorial library. The invention thus also provides in certain embodiments an agent capable of altering mitochondrial permeability transition, wherein the agent is identified by the methods just described. In certain other embodiments the invention provides a method for altering survival of a cell, comprising contacting a cell with an agent identified according to the methods just described, under conditions and for a time sufficient to modulate cell survival. In certain other embodiments, the invention provides a method for altering mitochondrial permeability transition (MPT), comprising contacting a mitochondrion with an agent identified according to the methods just described, under conditions and for a time sufficient to alter MPT. In certain further embodiments the mitochondrion is present within a cell. In certain further embodiments the cell is present within a living organism. In other embodiments the cell is a cybrid cell.
In still another aspect the present invention provides a method for preparing a mitochondrial permeability transition pore component polypeptide fused to an energy transfer molecule, comprising the steps of (a)culturing a host cell comprising a nucleic acid expression construct that encodes a fusion protein comprising an adenine nucleotide translocator polypeptide or a derivative thereof fused to an energy transfer molecule polypeptide or a derivative thereof, under conditions that permit expression of the fusion protein; and (b) recovering the fusion protein from the culture. In certain embodiments the mitochondrial permeability transition pore component is an adenine nucleotide translocator, which in certain further embodiments is human ANT1, human ANT2 or human ANT3. In certain other embodiments the mitochondrial permeability transition pore component is porin, hexokinase, creatine kinase, PRAX, CAML or the peripheral benzodiazepine receptor. In another embodiment the invention provides a method for preparing a cyclophilin polypeptide fused to an energy transfer molecule, comprising the steps of (a) culturing a host cell comprising a nucleic acid expression construct that encodes a fusion protein comprising a cyclophilin polypeptide or a derivative thereof fused to an energy transfer molecule polypeptide or a derivative thereof, under conditions that permit expression of the fusion protein; and (b) recovering fusion protein from the culture. In certain embodiments the cyclophilin polypeptide is a cyclophilin D polypeptide, and in certain other embodiments the cyclophilin polypeptide is human cyclophilin A, cyclophilin B, human cyclophilin C or human Cyp-60. In certain embodiments the host cell is a prokaryotic cell, and in certain other embodiments the host cell is a eukaryotic cell, which in certain further embodiments is a 293, a COS-1, a COS-7, a Sf9, a CHO, a Hep-2, a MDCK or a Jurkat cell. In other embodiments the nucleic acid expression construct is extrachromosomal. In other embodiments the nucleic acid expression construct is integrated into a host cell chromosome, which in certain further embodiments is a mitochondrial chromosome. In certain other embodiments the fusion protein comprises a recognition sequence for a protease, and in certain other embodiments the fusion protein comprises a ligand for a receptor.
In certain other embodiments the invention provides a kit for screening for agents that alter mitochondrial permeability transition, comprising (a) an isolated cyclophilin D polypeptide or a derivative thereof; (b) an isolated adenine nucleotide translocator polypeptide or a derivative thereof; and (c) a detection reagent that specifically binds to at least one of the foregoing polypeptides. In certain embodiments the cyclophilin D polypeptide is immobilized on a support. In certain other embodiments the adenine nucleotide translocator polypeptide is immobilized on a support. In other embodiments the detection reagent is an antibody or antigen-binding fragment thereof. In another embodiment the invention provides a kit for screening for agents that alter mitochondrial permeability transition (MPT), comprising (a) a host cell; (b) a first nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding an adenine nucleotide translocator polypeptide fused to a first energy transfer molecule or a variant thereof; and (c) a second nucleic acid expression construct, comprising a promoter operably linked to a polynucleotide encoding a cyclophilin D polypeptide fused to a second energy transfer molecule or a variant thereof. In a further embodiment the host cell is a prokaryotic cell, and in a different further embodiment the host cell is a eukaryotic cell, which in certain further embodiments is a 293, a COS-1, a COS-7, a Sf9, a CHO, a Hep-2, a MDCK or a Jurkat cell. In certain other embodiments, the first and second energy transfer molecules are green fluorescent protein (GFP), blue-shifted GFP, cyan-shifted GFP, red-shifted GFP or yellow-shifted GFP.
These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entireties as if each was incorporated individually.